NZ522733A

NZ522733A - Non-antibacterial tetracycline as anti-fungal agents

Info

Publication number: NZ522733A
Application number: NZ522733A
Authority: NZ
Inventors: Yu Liu; Lorne M Golub; Maria Ryan; Hsi-Ming J Lee; Sanford Simon
Original assignee: Yu Liu; Lorne M
Priority date: 2000-06-02
Filing date: 2001-06-01
Publication date: 2004-05-28
Also published as: KR20030024683A; CA2411342A1; EP1296703A4; EP1296703B1; DE60108873D1; AU2001268135A1; JP2003535140A; EP1296703A1; ATE288779T1; WO2001093891A1

Abstract

The specification relates to a method of inhibiting growth of a fungus in a non-human mammal. The method involves contacting the fungus with an effective amount of chemically-modified tetracycline compound (CMT) selected from the group consisting of CMT-3, CMT-4, CMT-7, CMT-8, CMT-315 and combinations of these. The fungus may be: Cryptococcus species, Candida albicans, Rhizopus species, Aspergillus fumigatus, Penicillium species, Absidia species, Scedosporium apiospermum, Phialophora verrucosa, Cunninghamella species, Tricothecium species, Ulocladium species or Fonsecae species. The chemically-modified tetracycline compound can be used for inhibiting the growth of a fungus in a mammal when prepared as a medicament and maybe used topically, by systemic administration or as a mouthwash.

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 522733 52273 1 NON-ANTIBACTERIAL TETRACYCLINE AS ANTI-FUNGAL AGENTS BACKGROUND OF INVENTION The invention relates to methods of inhibiting the growth of fungus. More specifically, the method relates to the use of non-antibacterial tetracycline compounds for the inhibition of fungal growth in a non-human mammal. Certain tetracycline compounds, including tetracycline itself, as well as sporocycline, etc., are broad spectrum antibiotics, having utility against a wide variety of bacteria. The parent compound, tetracycline, has the following general structure: Hie numbering system for the multiple ring nucleus is as follows: Tetracycline, as well as the 5-OH (terramycin) and 7-C1 (aureomycin) derivatives, exist in nature^ and are all well known antibiotics. Semisynthetic derivatives such as 7-dimethylamino-tetracycline (minocycline) and 6a-deoxy-5- Tntellectual property office of n.z 2 FEB 2004 Dcrci\/pn WO 01/93891 PCT/US01/17750 2 hydroxy-tetracycline (doxycycline) are also known antibiotics. Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained to do so. A class of compounds has been defined that are structurally related to the 5 antibiotic tetracyclines, but which have had their antibiotic activity substantially or completely expunged by chemical modification. Modifications to the basic tetracycline structure have been reviewed by Mitscher (1978). According to Mitscher, modification at positions 5-9 of the tetracycline ring system can be made without causing the complete loss of antibiotic properties. 10 However, changes to the basic structure of the ring system, or replacement of substituents at positions 1-4 or 10-12, generally lead to synthetic tetracyclines with substantially less, or essentially no, antibacterial activity. For example, 4-de(dimethyIamino)tetracycline is commonly considered to be a non-antibacterial tetracycline. These compounds, known as chemically-modified tetracyclines (CMTs), 15 have been found to possess a number of interesting properties, such as the inhibition of excessive mammalian collagenolytic activity both in vitro and in vivo. See, for example, Golub et al. (1991). It has been established that tetracyclines, which are rapidly absorbed and have a prolonged plasma half-life, exert biological effects independent of their 20 antibacterial activity (Golub et al. 1991, Golub et al. 1992, Uitto et al. 1994). Such effects include inhibition of some but not all animal and human derived matrix metalloproteinases. Studies have suggested that, in some systems, certain tetracyclines and inhibitors of metalloproteinases can inhibit tumor progression (DeClerck et al. 1994) 25 or angiogenesis (WIPO publication WO 92/12717; Maiagoudakis et al. 1994). Zucker et al. (1985) showed that minocycline can inhibit melanoma cell activity in vitro. Some tetracyclines may exhibit cytostatic effects against some tumors (Kroon et al. 1984; van den Bogert et al. 1986). Pro-gelatinase A (MMP-2) has been reported to be associated with tumor spread (Yu et al. 1997). 6-demethyl-6-deoxy-4-30 de(dimethylamino)tetracycline (CMT-3) has been shown to suppress prostate and 3 melanoma tumor growth and metastasis in vivo (Lokeshwar et al. 1998; Seftor et al. 1998). Fungal growth is an important clinical problem, especially in patients whose immune system has been depressed. There are relatively few anti-fungal drugs 5 presently available for clinical use. Additionally, fungi are developing resistance to the few anti-fungal agents approved for clinical use. Currently available anti-fungal agents are generally quite toxic and can cause side effects such as renal impairment, phlebitis, and gastrointestinal disturbance. Some anti-fungal agents, such as Nystatin™ are poorly absorbed by the 10 gastrointestinal tract Thus, there is a need for new and improved anti-fungal agents. SUMMARY OF THE INVENTION It has now been discovered that these and other objectives can be achieved by the present invention, which provides a method for inhibiting fungal growth in a non-human mammal in need thereof by administering a tetracycline, preferably a chemically 15 modified tetracycline (CMT), in an amount that is effective to inhibit the growth of fungus but that has substantially no antibacterial activity. DETAILED DESCRIPTION OF INVENTION The present invention provides a method of inhibiting the growth of fungus. The method includes the administration of a tetracycline compound that is effective in 20 inhibiting the growth of fungus, but has substantially no antibacterial activity. Fungus as defined herein includes any eukaryotic single celled organism characterized by the absence of chlorophyll and by the presence of a rigid cell wall. Preferred fungi are clinically significant fungi, meaning such fungi grow in or on mammal s. Examples of clinically significant fungi include Cryptococcus species, 25 Candida albicans, Rhizopus species, Aspergillus fumigatus, Penicillium species, Absidia species, Scedosporium apiospermum, Phialophora verrucosa, Cimninghamella species, Tricothecium species, Ulocladium species, and Fonsecae species. " :NTELLECTUAL PROPERTY OFFICE OF N.Z 2 <t FEB 20M Dcr>ciwer> WO 01/93891 PCT/USO1/17750 4 Tetracycline compounds utilized in the method of the invention can inhibit the growth of more than one type of fungus. In addition, some tetracycline compounds inhibit the growth of some species of fungi more effectively than others. Accordingly, a single tetracycline compound or combination of tetracycline 5 compounds can be administered to inhibit the growth of a plurality of fungi. The tetracycline compound administered can be an antibacterial or non-antibacterial compound or a combination thereof. The tetracycline compound has the general structure indicated above. Some examples of antibacterial tetracycline compounds include doxycycline, 10 minocycline, tetracycline, oxytetracycline, chlortetracycline, demeclocycline, lymecycline and their pharmaceutical^ acceptable salts. Doxyclycline is preferably administered as its hyclate salt or as a hydrate, preferably monohydrate. The antibacterial tetracycline compound is administered in an amount that is effective in reducing the growth of fungus, but does not significantly reduce the 15 growth of bacteria. For example, tetracycline compounds that have significant antibacterial activity may be administered in an amount which is 10-80% of the antibacterial amount. More preferably, the antibacterial tetracycline compound is administered in an amount which is 40-70% of the antibacterial amount. 20 Some examples of antibacterial amounts of members of the tetracycline family include lOOmg/day of doxycycline, lOOmg/day of minocycline, 250mg of tetracycline four times a day, lOOOmg/day of oxytetracycline, 600mg/day of demeclocycline and 600mg/day of lymecycline. In a preferred embodiment, a non-antibacterial tetracycline is administered. 25 Non-antibacterial tetracycline compounds are structurally related to the antibacterial tetracyclines, but have had their antibiotic activity substantially or completely eliminated by chemical modification. Chemically modified tetracyclines are referred to herein as CMTs. WO 01/93891 PCT/US01/17750 For example, non-antibacterial tetracycline compounds are capable of achieving antibacterial activity comparable to that of tetracycline at concentrations at least about ten times, preferably at least about twenty five times, greater than that of tetracycline. 5 It has been discovered that tetracyclines having substantially no antibacterial activity can exhibit anti-fungal activity. Antibiotics, such as tetracyclines, are known to inhibit the growth of bacteria. However, it was generally believed that antibiotics, such as tetracyclines, in therapeutic doses, were unable to destroy nucleated (eukaryotic) cells such as fungus. It has now been discovered that tetracyclines 10 having little or no antibacterial activity, such as CMTs, can exhibit excellent antifungal acitivity, while demonstrating low cytotoxicity. Examples of chemically modified non-antibacterial tetracyclines (CMTs) include those that lack the dimethylamino group at position 4 of the tetracycline ring structure, e.g.,: 15 4-de(dimethylamino)tetracycline (CMT-1), 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT -3 ), 7-chloro-4-de(dimethylamino)tetracycline (CMT-4), 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), 4-de(dimethylamino)-12a-deoxytetracycline (CMT-7), 20 6-deoxy-5a-hydroxy-4-de(dimethylamino)tetracycline (CMT-8), 4-dedimethylamino-12a-deoxyanhydrotetracycline (CMT-9), 7-dimethylamino-6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-10), 4-de(dimethylamino)-5-oxytetracycline, 25 5a,6-anhydro-4-hydroxy-4'-de(dimethyIamino)tetracycline, 4-de(dimethylamino)-ll-hydroxy-12a-deoxytetracycline, 12a-deoxy-4-deoxy-4-de(dimethylamino)tetracycline, and 12a,4a-anhydro-4-de(dimethylamino)tetracycline. 30 Further examples of tetracyclines modified for reduced antibacterial activity include 6-a-benzylthiomethylenetetracycline, the mono-N-alkylated amide of 01/22/2002 THE 14:54 FAX 5168223582 HOFFMANN&BARON ® 00; 6 PCT/US01/17750 IPEA/US 2 2 JAN 2002 tetracycline, 6-fluoro-6-demethyltetracyclir c, 11 a-chlorotetracycline, letracyclinonitrile (CMT-2), and tetracycline pyrazole (CMT-5). The preferred chemically-modified Tetracyclines include CMT-3, CMT-4, CMT-7 and CMT-8. CMT-3, CMT-7, and 'SMT-8 art: most preferred. Derivatives of CMTs as mentioned <ibove can also be used. Derivatives of CMTs include any compound derived from a CMT. Combinations of tetracycline compounds, such as CMTs and/or their derivatives, can be used in the method of the invention. CMTs in combination with other tetracyclines and/or pharmaceutical compounds can also be employed. For example, derivatives of CMT-3 include: CMT-3 01 7-BROMO CMT-3 02 7-NITRO CMT-3 03 9-lsITRO CMT-3 04 7-ACETAMEDO CMT-3 05 9-ACETAMIDO CMT-3 06 9-DTMETHYLAMTNO CMT-3 07 7-AMINO CMT-3 08 9-^yvlINO CMT-3 09 9-DIMETHYLAMINQACETAM1DO CMT-310 7-DJMJiTHYLAMlNO CMT-311 9-PALM1TAMIDE CMT-312 2-CONHCH2-PYROLIDIN-l -YL CMT-3 13 2-CONHCHj-PIPERIDIN-l-YL CMT-314 2-CONHCH, -MORPHOLIN-1 -YL CMT-315 2-CONHCH2-PIPERAZIN-l-YL derivatives of CMT-8 includc: f CMT-8 01 9-ACETAMIDO ! CMT-802 9-DIMETHYLAMINOACETAMIDO . CMT-803 9-1 'ALM3TAMIDE j CMT-S04 9-ITITRO CMT-805 9-JVMINO CMT-8 06 9-DIMETHYLAMINO CMT-S07 2-CONHCH2-PYRROr.TDTN-l -Y1 CMT-S08 2-CONHCH,.PIPERlDIN-l-YL CMT-8 09 2-CONHCH,-PI?ERAZTN-l-YL AMENDED SHEET 01/22/2002 TUE 14:54 FAX 5168223582 HOFFMANN&BARON [2)006 Derivatives of CMT-10 include: PCWJS01/17750 IPEA/US 2 2 JAN 2002 CMT-lOOl 7- DIMETHYLAMINOS ANCYCL1NE CMT-1002 9-NHTRO Preferred derivatives are CMT-302, CMT-306, CMT-308 and CMT-315. CMT-308 and CMT-315 are most preferred. 5 .Further examples of such CMT derivatives include the structures described in U.S. Appl. Serial No. 09/573,654 assigned to CoIlaGcnex Pharmaceuticals, Inc. of Newtown, PA, which application and compounds described therein is incorporated herein by reference in its entirety. The tetracycline compounds administered can be in the form of 10 pharmaceutically acceptable salts. Thetenr "pharmaccutically acceptable salt" refers to a salt prepared from tetracycline compounds and pharmaceutically acceptable -nontoxic acids or bases. The acids may be inorganic or organic acids of tetracycline compounds. Examples of inoTganic acids include hydrochloric, hydTobroxnic, hydroiodic, sulfuric, and phosphoric acids. Examples of organic acids include 15 carboxylic and sulfonic acids. Hie radical of the organic acids may be aliphatic or aromatic. Some examples of organic acids include formic, acetic, phenylacetic, propionic, succinic, glycolic, glucuronic, mslcic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (patnoic), methanesulfonic, ethanesulfonic, panthenoic, benzenesulfonic, stearic, sulfarulic, alginic, tartaric, citric, 20 gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriate organic bases may be selected, for example, from N,N-dibenzyletliylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-raethylglucamine), and procaine. Suitable inorganic bases include sodium hydroxide and potassium hvdroxide. 25 The inhibition of fungal growth occurs over a wide range of concentrations. The effective amount of tetracycline used according to the invention is an amount that is effectively inhibitory of fungal growth activity. An amount of tetracycline is effectively inhibitory to fungal growth activity if it significantly reduces fungal AMENDED SHEET WO 01/93891 PCT7US01/17750 8 growth activity or fungal growth. Inhibition of fungal growth can also include the inhibition of the invasiveness of the fungus, for example, in mammalian tissue. The minimal amount of tetracycline administered is the lowest amount capable of inhibiting or eliminating the growth of fungus. CMTs exhibit their anti-fungal 5 inhibitory properties at concentrations that lead to relatively few side effects, and in some cases are substantially free of side effects. In general, CMTs can be used at higher levels than antibacterial tetracyclines, while avoiding the disadvantages associated with antibacterial activity, such as the indiscriminate killing of beneficial bacteria, and the emergence of resistant bacteria which often accompanies the use of 10 antibacterial compounds over prolonged periods of time. Preferably, the CMT has a low phototoxicity, or is administered in an amount that results in a serum level at which the phototoxicity is acceptable. Phototoxicity is a chemically-induced photosensitivity. Such photosensitivity renders skin susceptible to damage, e.g. sunburn, blisters, accelerated aging, erythemas and eczematoid 15 lesions, upon exposure to light, in particular ultraviolet light. Phototoxicity can be evaluated in terms of a photoiiritancy factor (PEF), as described in the examples. A PIF value of about 1.0 indicates that a compound is considered to have no measurable phototoxicity. The low phototoxic derivatives preferably have PIF values no greater than 20 about 5, preferably no greater than about 2, more preferably no greater than about 1.5, most preferably no greater than about 1.2, and optimally about 1. An example of a CMT having a low phototoxicity is CMT-8. The CMTs useful according to the invention possess a desirable but unusual combination of physicochemical properties, including activity, bioavailability, 25 solubility, and reduction of side effects. These properties render the compounds particularly desirable for the inhibition of fungal growth activity in mammals. In addition, it is believed that the properties of hydrophilicity and hydrophobicity are well balanced in these compounds, enhancing their utility both in vitro and especially in vivo, while other compounds lacking such balance are of substantially less utility. 30 Specifically, the compounds have an appropriate degree of solubility in aqueous WO 01/93891 PCT/US01/17750 9 media to permit absorption and bioavailability in the body, while also having a degree of solubility in lipids to permit traversal of the cell membrane to a putative site of action. CMT-3, which is highly lipophilic, may be readily absorbed by tissues during topical administration. The compounds are maximally effective if they can be 5 delivered to the site or region of the fungal growth activity. In the treatment of certain localized conditions, the degree of hydrophilicity of the CMT can be of lesser importance. Such compounds as tetracyclinonitrile (CMT-2) and 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), which have low solubility in aqueous systems, can be used in direct or topical treatment of fungal 10 growth. Animal experiments, in which adult rats are orally gavaged with these two CMTs, have shown no detectable blood levels of these compounds, indicating a lack of systemic absorption and/or extraordinarily rapid excretion. The method of the invention can be used wherever the growth of fungus is to be diminished or eliminated, including in vitro, ex vivo, or in vivo. In vitro systems 15 typically include cultured samples. Ex vivo biological systems typically include cells or organ systems removed from a living animal. In vivo uses are limited to biological systems that are living, and such uses typically include therapeutic or pharmaceutical interventions. Thus, embodiments of the invention in which a tetracycline compound is administered to a mammal are representative of in vivo methods. 20 The method of the invention is useful to treat medical conditions characterized by fungal growth activity. In particular, the invention is useful in the treatment (e.g., palliation, amelioration) of medical conditions characterized by excessive or pathological levels of fungal growth activity. Such conditions are often caused by a depressed immune system. For example, AIDS patients or cancer patients undergoing 25 chemotherapy often suffer from development of fungal growth in the mouth and other areas of the body. Accordingly a method of treating fungal growth in a living organism in need thereof is specifically provided. The method includes administering to the living organism tetracycline in an amount that is effective in inhibiting the growth of fungus, 30 but has substantially no antibacterial activity. WO 01/93891 PCT/USO1/17750 10 In a preferred embodiment, the tetracycline is a CMT. Preferred CMTs include CMT-3, CMT-4, CMT-7, CMT-8, CMT-302, CMT-306, CMT-308, and CMT-315. CMT-3, CMT-7, CMT-8, CMT-308, and CMT-315 are most preferred. Administration can be topical or systemic. Effective amounts can be readily 5 determined by those skilled in the art. In a preferred embodiment, the living organism is a mammal. Mammals include, for example, humans, baboons and other primates, as well as pet animals such as dogs and cats, laboratory animals such as rats and mice, and farm animals such as horses, sheep, and cows. Humans are preferred. 10 Living organisms in need of inhibition of fungal growth can also include plants. Plants include for example crop plants, such as wheat, barley, oats, soybeans, cotton; vegetables; fruit trees and plants; and ornamental plants. Screening for antifungal CMTs with SABHI Agar culture showed that CMT-315, CMT-3, CMT-7 and CMT-308 are highly effective in inhibiting the growth of 15 various tested fungi, including Penicillium species, Aspergillus fumigatus, Rhizopus species, and Candida albicans (Table 1 and 2). For example, CMT-315 significantly inhibited the growth of the four fungi including Rhizopus sp., which is the fastest growing fungus in culture. The significance of this finding is that all these four tested fungi can cause human diseases. 20 For example, Penicillium species are responsible for pulmonary infection, skin infection, external otomycosis, mycotic keratitis, endocarditis, and cutaneous ulceration; Aspergillus fumigatus can cause allergic bronchopulmonary infection, fungus ball, invasive pulmonary infection, skin and nail infection, osteomyelitis, external otomycosis, mycotic keratitis, sinusitis, myocarditis, renal infection and brain 25 abscess; Rhizopus species cause Rhinocerebral infection, pulmonary infection, mycotic keratitis, intraocular infeciton, orbital cellulitis, deep wound infection, external otomycosis, dermatitis, etc. These fungi usually exist in air, soil, food, surfaces such as furniture, and sometimes in human body, such as in the mouth. The fungi enter tissues of living WO 01/93891 PCT/USO1/17750 11 organisms and cause infection, especially in mammals when the immune system becomes weaker, such as in patients having diabetes, AIDS, cancers, transplants, etc. The invention can also be practiced by including with the tetracycline compound one or more other therapeutic agents, such as any conventional anti-fungal 5 agent. The combination of the tetracycline compound with such other agents can potentiate the therapeutic protocol. Numerous therapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. The preferred pharmaceutical composition for use in the method of the 10 invention includes a combination of the tetracycline compound in a suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the art. Examples of carriers and excipients include starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols. 15 The tetracycline compound may be administered to mammals by sustained release, as is known in the art. Sustained release administration is a method of drug delivery to achieve a certain level of the drug over a particular period of time. The level typically is measured by serum concentration. Further description of methods of delivering tetracycline compounds by sustained release can be found in U.S. Serial 20 Application No. 60/281,854 and assigned to CollaGenex Pharmaceuticals, Inc. of Newtown, Pennsylvania, which application incorporated herein by reference in its entirety. Systemic administration can be enteral or parenteral. Enteral administration is a preferred route of delivery of the tetracycline, and compositions including the 25 tetracycline compound with appropriate diluents, carriers, and the like are readily formulated. Liquid or solid (e.g., tablets, gelatin capsules) formulations can be employed. 30 Parenteral use (e.g., intravenous, intramuscular, subcutaneous injection) is also contemplated, and formulations using conventional diluents, carriers, etc., such as are known in the art can be employed to deliver the compound. WO 01/93891 PCT/US01/17750 12 Alternatively, delivery of the tetracycline compound can include topical application. Compositions deemed to be suited for such topical use include as gels, salves, lotions, ointments and the like. In a preferred embodiment, the tetracycline compound is included in a mouthwash. 5 Administration to plants can be performed by any known means. Such means include, for example, application by spray or dust to the surface of the plant or the surrounding soil. The maximal dosage for a mammal is the highest dosage that does not cause undesirable or intolerable side effects. Such doses can be readily determined by those 10 skilled in the art. For example, CMTs can be systemically administered in a mammal in an amount of from about O.lmg/kg/day to about 60mg/kg/day, and preferably from about lmg/kg/day to about 18mg/kg/day. The practitioner is guided by skill and knowledge in the field, and the present invention includes, without limitation, dosages that are effective to achieve the desired anti-fungal activity. 15 The appropriate dose of the tetracycline compound for topical administration can also be readily determined by those skilled in the art. For example, topical administration of CMTs in amounts of up to about 25% (w/w) in a vehicle can be administered without any toxicity in a human. Amounts from about 0.1 % to about 10% are preferred. 20 Most anti-fungal drugs presently available in clinics are much more toxic to mammals, such as humans, than antibacterial drugs. For example, amphotericin B may not only cause disorders of the digestion system, but also cause damage to the nervous system. CMTs are usually less toxic to mammals than conventional anti-fungal 25 compounds. For example, CMT-3 is being tested in cancer patients without evidence of side effects, except at unusually high doses. Also, CMT's are often absorbed into the system better than conventional anti-fungal drugs. Particular CMTs have only limited biodistribution, e.g. CMT-5. In such cases, topical administration is the preferred route of administration of the compound. WO 01/93891 PCT/US01/17750 13 The following examples are provided to assist in a further understanding of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope thereof. 5 EXAMPLE 1 The following example demonstrates a response of selected fungi to CMT-3, 4, 7, 8, and the following derivivatives of CMT-3: 302, 303, 306, 308, 309 and 315. The following fungi were inoculated onto potato dextrose agar (PDA) from stock cultures and incubated aerobically at 30°C: Aspergillus fumigatus ATCC 1022, 10 Penicillium sp. (laboratory isolate), Candida albicans, ATCC 14053, and Rhizopus sp. A sterile cotton tipped applicator was moistened with sterile 0.9% saline and rolled over the surface of PDA slants of Aspergillus fumigatus, Rhizopus sp. and Penicillium sp. which demonstrated copious conidiogenesis. The conidia were 15 suspended in 0.9% saline and the turbidity was adjusted to match a 0.5 MacFarland standard (equivalent to approximately 1.5 x 10s cells). Candida albicans was suspended in saline and adjusted to 0.5 MacFarland in a similar manner. These suspensions were diluted 1:100 in sterile 0.9% saline. SABHI Agar (Difco) pH 7.0 was prepared in 100ml amounts and sterilized at 20 121°C for 15 min. After the SABHI agar base cooled to 50°, 10 ml of each of the CMT substances were prepared in 10% DMSO at a concentration of 250 p.g/ml. The CMT substances were than added at a final concentration of 25 (J.g/ml of agar base. SABHI Agar plates of each CMT and SAHBI agar without CMT using dimethylsulphoxide (DMSO) as a control were inoculated with IOjjI of conidia 25 suspension of Aspergillus fumigatus, Penicillium sp. and Rhizopus sp. and 10p.l suspension of Candida albicans prepared as described above. The plates were then incubated aerobically for 24 hour and for 48 hours at 30°C. WO 01/93891 PCT/USO1/17750 14 The results are set forth in Table 1 (24hr. incubation) and Table 2 (48 hr. incubation). The score table used for Tables 1 and 2 is set forth in Table 3. Table 1 Growth at 25 jag/ml compared to control at 24 hrs incubation Organism 3 4 7 8 302 303 306 308 309 315 DMSO Aspergillus Fumigatus 0 0 0 ± 0 ± 1 0 ± 0 3 Penicillium Sp. 0 3 3 3 3 3 3 0 3 0 4 Rhizopus sp. 3 4 4 4 4 4 4 4 4 1 4 Candida Albicans 1 1 0 4 4 3 3 4 4 0 4 5 Table 2 Growth at 25|xg/ml compared to control at 48 hours Organism 3 4 7 8 302 303 306 308 309 315 DMSO Aspergillus Fumigatus 4 1 4 4 4 4 4 1 4 0 4 Penicillium Sp. 4 0 4 4 4 4 4 0 4 0 4 Rhizopus sp. 1 4 3 4 4 4 4 4 4 1 4 Candida Albicans 4 4 0 4 4 4 4 4 4 0 4 10 Table 3 Inhibition Score and Grading of Fungal Growth Growth Grade Inhibition Score Description 4 0% Level of growth in the absence of 15 anti-fungal agent (control). 3 25% 25% reduction in growth of colonies compared to control. WO 01/93891 15 PCT/US01/17750 50% 75% 100% 50% reduction in growth of colonies compared to control. 75% reduction in growth in colonies compared to control. complete inhibition of growth. 10 CMT-315 yielded the best results with activity against all the fungi tested. CMT-308 demonstrated activity against Aspergillus fumigatus and Penicillium sp.. CMT-4 demonstrated activity against Penicillium sp., and Aspergillus f. CMT-7 demonstrated strong activity against Candida albicans. CMT-3 inhibited Rhizopus sp., which is the most rapidly growing of the fungi, and can cause Rhiaocerebral 15 infection, pulmonary infection, mycotic keratitis, intraocular infection, orbital cellulitis, deep wound infection, external otomycosis, dermatitis, etc. EXAMPLE 2 This example demonstrates a direct comparison between CMT-3 and Amphotericin B (AmB), a conventional anti-fungal agent, in the inhibition of 20 Aspergillus f. The plates were prepared as described above, using 0.125, 0.5, 0.50, 1.00 and 2.00 concentrations of each of the drugs tested. DMSO was used as a control The results are shown in Table 4 below. The results were graded according to the criteria set forth in Table 3. 25 Table 4 Cone. (p,g/ml) 0.125 0.25 0.50 1.00 2.00 CMT-3 4 2 1 ±0 0 AmB 4 2 1 0 0 The results demonstrate that at various concentrations, the CMT-3 inhibited growth of Aspergillus f as effective as AmB. At a concentration of 1.0 (ig/ml, AmB inhibited 100% of fungal growth, while CMT-3 inhibited 95% of growth. At 2.0 WO 01/93891 PCT/USO1/17750 16 |ig/ml, both AmB and CMT-3 inhibited 100% of growth. Importantly, unlike AmB, CMT-3 demonstrates very little toxicity in vivo at 2.0 ng/ml concentration. EXAMPLE 3 This example demonstrates the concentration of anti-fungal agent required to 5 reduce the growth of the fungus by 50% in vitro (IC50) and the minimum concentration required to completely inhibit the growth of the fungus in vitro (MIC). CMTs utilized in the method of the invention, i.e CMT-3 and CMT-8 were compared to Doxycycline and Amphotericin B on microplate agar gels. Each drug was dissolved in DMSO (1.0 mg/ml) as a stock solution and stored 10 at -20°C. Just prior to use, each stock solution was thawed and diluted in DMSO to produce 6 different lOOx concentrations. Potato dextrose agar was dissolved in distilled water (39 g/L) and sterilized at 138°C (250°F) for 15 min. The agar solution was mixed with each drug (in a water bath at 60°C) to make a series of final concentrations, i.e. 0.00, 0.25, 0.50, 1.00, 2.00, 4.00 |ig/ml. The mixtures were then 15 transferred to 24-well plates (1 ml/well). After the gel had formed, the fungus in PBS (spore count = l-5xl04/ml) was inoculated by pipetting lOpl onto each gel. The plates were incubated at 30°C for different times, depending on the requirement of each species, e.g. 24 hours for Penicillium, Rhizopus, Tricothecium, Ulocladium, Absidia, Aspergilus, Candida, Cunninghamella, 3 days for Scedosporium, and 5 days 20 for Fonsecae and Phialophora. The MICs and IC50s for the 11 different fungi are set forth in Table 5. indicates better than or similar results to Amphotericin B. "NI" indicates no detectable inhibition. Table 5 25 IC50fag/mD Candida Albicans AmB 0.5 CMT-3 1.0 30 CMT-8 NI Doxy NI Rhizopus Species MICfug/ml) 1.0 2.0 NI NI WO 01/93891 PCT/US01/17750 17 AmB 0.4 1.0 CMT-3 0.8 2.0 CMT-8 NI NI Doxy NI NI 5 Aspergilus Fumigatus AmB 0.8 2.0 CMT-3 0.5 1.0* CMT-8 NI NI 10 Doxy NI NI Penicillium Species AmB 0.12 0.25 CMT-3 0.2 0.5 15 CMT-8 2.0 >4 Doxy NI NI Absidia Species AmB 1.0 4.0 20 CMT-3 1.5 4.0* CMT-8 NI NI Doxy NI NI Scedosporium Apiospermum 25 AmB 4.0 >4 CMT-3 0.2 1.5* CMT-8 2.0 >4 Doxy NI NI 30 Phialophora Verrucosa AmB NI NI CMT-3 1.5 4.0* CMT-8 NI NI Doxy NI NI 35 Cunnuisrhamella Species AmB NI NI CMT-3 2.0 4.0* CMT-8 NI NI 40 Doxy NI NI Tricothecium Species AmB NI NI CMT-3 0.2 1.5* 45 CMT-8 0.7 2.0 Doxy 4.0 >4 WO 01/93891 18 PCT/US01/17750 Ulocladium Species AmB CMT-3 CMT-8 1.0 0.25 2.0 NI 2.0 1.0* >4 NI 5 Doxy Fonsecae Species 10 CMT-8 Doxy AmB CMT-3 4.0 1.0 NI NI >4.0 4.0* NI NI Thus, CMT-3 was effective on all 11 tested fungi, and CMT-8 had effects on some of these fungi. However, for 8 fungi out of the 11 different species of fungi, 15 Amphotericin B showed the same or less antifungal activity than CMT-3. Doxy had essentially no detectable antifungal activity in this experiment. This example demonstrates the antifungal activity of CMT-3 and Amphotericin B in vitro as being fungistatic (i.e. arresting the growth of the fungus) 20 or fungicidal (i.e. killing the fungus). In the pre-treatment phase of the experiment, Penicillium spores were suspended in PBS to achieve a spore count of 107/ml. CMT-3 and Amphotericin B were dissolved in DMSO to reach a concentration of 1.0 mg/ml as stock solutions. 10 or 50 (j,l aliquots of these stock solutions were added to the incubation mixture 25 (containing 1.0 ml of 107/ml of Penicillium spores in PBS) to achieve a final concentration of 10 |o,g/ml or 50 |i.g/ml, respectively, for both drugs. The various incubations of Penicillium were carried out for 24 hours at 30°C. After the pre-treatment phase, the reaction mixtures were diluted 1000 times with PBS, reducing the concentration of both drugs to 0.01 ng/ml or 0.05 iig/ml, and 30 reducing the Penicillium spore count to 104/ml. These drug concentrations of both CMT-3 and Amphotericin B would not be expected to inhibit the growth of the viable Pencillium spores. EXAMPLE 4 Controls were then prepared. Before incubation, each tube was either not diluted further, or diluted to Vz or lA with PBS to produce tubes with three different WO 01/93891 PCT/US01/17750 19 spore counts, ie, 104/ml, or 0.5 x 104/ml, or 0.25 x 104/ml. These cultures were then inoculated on potato dextrose agar gels in 24-well plates, and incubated at 30°C for 48 hours to determine the rate of growth of the fungus as described before. The controls were prepared from the suspension in the pre-treatment phase 5 containing only Penicillium spores 107/ml, and PBS. This control was diluted by 1000 times with PBS to produce a spore count of 104/ml. 1.0 ml of this diluted spore suspension was added to eight tubes. The stock solutions of CMT-3 and Amphotericin B, and DMSO were also diluted by 1000 times with PBS (the new concentration being 1.0 (ig/ml for both drugs and 0.1% for DMSO), and 10 or 50 jj! of these 10 solutions was added into the above tubes. The final concentrations in each tube was either 0.01 (xg/ml or 0.05 |xg/ml for both drugs (CMT-3 or AmB), or 0.001% or 0.005% for DMSO. These tubes were further treated as described above to determine the growth of the fungus as controls. The results demonstrated that all controls, including Hie concentration of 0.01 15 and 0.05 p.l/ml of both drugs (CMT-3 and Amphotericin B), showed the same growth rate of Penicillium as the cultures without drugs, demonstrating that these low concentrations of both drugs did not inhibit the growth of the fungus in these control cultures. Cultures of the Penicillium, after pretreatment with 10 and 50 jil/ml of 20 Amphotericin B, showed the same rate of growth as PBS and DMSO controls during the subsequent incubation phase of the experiment, indicating that this drug did not kill the spores during the pre-treatment phase. In contrast, cultures of Penicillium after pretreatment with 10 and 50 |il/ml of CMT-3, showed little or no growth on the agar gels compared with the controls, 25 demonstrating that CMT-3 did kill the fungal spores during the pre-treatment phase. Thus, Amphotericin B exhibited fungistatic activity, i.e. fungal growth was arrested but the fungal spores were not killed. On the other hand, CMT-3 exhibited fungicidal activity against Penicillium, killing the fungus. WO 01/93891 20 References PCT/USO1/17750 DeClerck YA, Shimada H, Taylor SM, and Langley KE, "Matrix metalloproteinases and their inhibitors in tumor progression," Annals NY Acad Sci 732:222-232 (1994). 5 Golub LM, Ramamurthy NS, McNamara TF, Greenwald RA, and Rifkin BR, "Tetracyclines inhibit connective tissue breakdown: New therapeutic implications for an old family of drugs," CritRev Oral Biol Med 2(2):297-322 (1991). Golub LM, Sorsa T, and Suomalainen K, Curr Opin Dent 2:80-90 (1992). Kroon AM, Dontje BHJ, Holtrop M, and van den Bogert C, "The 10 mitochondrial genetic system as a target for chemotherapy: tetracyclines as cytostatics," Cancer Letts 25(l):33-40 (1984). Lokeshwar BL, Selzer MG, Dudak SM, Block NL, and Golub LM, "Inhibition of tumor growth and metastasis by oral administration of a non-antimicrobial tetracycline analog (CMT-3) and doxycycline in a metastatic prostate cancer model," 15 Cancer Res (1998). Maragoudakis ME, Peristeris P, Missirlis E, Aletras A, Andriopoulou P, and Haralabopoulos G, Annals NY Acad Sci 732:280-293 (1994). Mitscher LA, The Chemistry of the Tetracycline Antibiotics, Ch. 6, Marcel Dekker, New York (1978). 20 Seftor REB, Sefitor EA, DeLarco JE, Kleiner DE, Leferson J, Stetler-Stevenson WG, McNamara TF, Golub LM, and Hendrix MJC, "Chemically-modified tetracyclines inhibit human melanoma cell invasion and metastasis," Clin Exp Metastasis 16 (1998). Uitto VJ, Firth JD, Nip L, and Golub LM, Annals NY Acad Sci 732:140-151 25 (1994). van den Bogert C, Dontje BHJ, Holtrop M, Melis TE, Romijn JC, van Dongen JW, and Kroon AM, "Arrest of the proliferation of renal and prostate carcinomas of </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO

01/93891 PCT/US01/17750 21 human origin by inhibition of mitochondrial protein synthesis," Cancer Res 46(7):3283-3289 (1986). Yu AE, Hewitt RE, Connor EW, Stetler-Stevenson WG, "Matrix metalloproteinases, Novel targets for directed cancer therapy," Clin Pharmacol 5 11:229-244(1997). Zucker S, Lysick RM, Ramamurthy NS, Golub LM, Wieman JM, and Wilkie DP, "Diversity of plasma membrane proteinases in mouse melanoma cells: Inhibition of collagenolytic activity and cytolytic activity by minocycline," J Natl Cancer Inst 75:517-525(1985). We Claim: 22 1. A method of inhibiting growth of a fungus in a non-human mammal in need thereof comprising contacting the fungus with an effective amount of a chemically-modified tetracycline compound (CMT) selected from the group consisting of CMT-3, CMT-4, CMT-7, CMT-8, CMT-308, CMT-315 and combinations thereof.
2. A method according to Claim 1 wherein said fungus is selected from the group consisting of Cryptococcus species, Candida albicans, Rhizopus species, Aspergillus fumigatus, Penicillium species, Absidia species, Scedosporivm apiospermum, Phialophora verrucosa, Cunninghamella species, Tricotkecium species, Ulocladium species, Fonsecae species, and combinations thereof.
3. A method according to Claim 1 "wherein said CMT is CMT-315.
4. A method according to Claim 1 wherein said fungus is selected from the group consisting of Rhizopus species, Absidia species, Scedosporivm apiospermum, Phialophora verrucosa, Cunninghamella species, Tricothecium species, Ulocladium species, Fonsecae species, or a combination thereof^ and wherein said CMT is CMT-3.
5. A method according to Claim 1 wherein said fungus is Aspergillus fumigatus, Penicillium species, Rhizopus species, Candida albicans, or a combination thereof, and wherein said CMT is CMT-315.
6. A method according to Claim 1 wherein said fungus is Penicillium species and said CMT is CMT-4.
7. A method according to Claim 1 wherein said fungus is Candida albicans and said CMT is CMT-7. N.,
8. A method according to Claim 1 wherein said fungus is Aspergillus fumigatus, Penicillium species or a combination thereof and said CMT is CMT-308. " rNTELLECTUAl"pROPERTY OFFICE OF N.Z 2 4 FEB 2004 RECEIVED 23
9. A method according to Claira 1 wherein said fungus is Penicillium specics, Scedosporium apiospermum, Tricothecitim species, Ulocladium species, Or a combination thereof and said CMT is CMT-8.
10. A method according to Claim 1 wherein said administration is systemic. A method according to Claim 10 wherein said CMT is administered in an amount from about 0.1 mg/kg/day to about (SO mg/kg/day. 1 12. A method according to Claim 10 wherein said CMT is administered in an amount from about 1 mg/kg/day to about 18 mg/kg/day. ,13. A method according to Claim 1 wherein said administration is topical. 14. A method according to Claim 13 wherein said CMT is administered in a mouthwash. 15. A use of a chemically-modified tetracycline compound (CMT) selected from the group consisting of CMT-3, CMT-4, CMT-7, CMT-8, CMT-308, CMT-315 and combinations thereof, in the manufacture of a medicament for inhibiting growth of a fungus in a mammal in need thereof. 16. A use according to claim 15 wherein said fungus is selected from the group consisting of Cryptococcus species, Candida albicans, Rhizopus species, Aspergillus fumigatus, Penicillium species, Absidia species, Scedosporium apiospermum, Phialophora verrucosa, Cunninghamella species, Tricothecium species, Ulocladium species, Fonsecae species, and combinations thereof. 17. A use according to claim 15 wherein said CMT is CMT-315. 18. A use according to claim 15 wherein said fungus is selected from the group consisting of Rhizopus species, Absidia species, Scedosporium apiospermum, Phialophora verrucosa, Cunninghamella species, Tricothecium species, Ulocladium species, Fonsecae species, or a combination thereof, and wherein said CMT is CMT-3. 19. A use according to claim 15 wherein said fungus is Aspergillus fumigatus, Penicillium species, Rhizopus species, Candida albicans, or a combination thereof, and wherein said CMT is CMT-315. I Intellectual property ippice of n.Z 2 h FEB 2004 24 20. A use according to claim 15 wherein said fungus is Penicillium species and said CMT is CMT-4. 21. A use according to claim 15 wherein said fungus is Candida albicans and said CMT is CMT-7. 22. A use according to claim 15 wherein said fungus is Aspergillus fumigatus, Penicillivm species or a combination thereof, and said CMT is CMT-308. 23. A use according to claim 15 wherein said fungus is Penicillium species, Scedosporium apiospermum, Tricothecium species, Ulocladium species, or a combination thereof and said CMT is CMT-8. 24. A use according to claim 15 wherein said mammal is a human. 25. A use according to claim 15 wherein the medicament is formulated for systemic administration. 26. A use according to claim 25 wherein the medicament is formulated to administer CMT in an amount from about 0.1 mg/kg/day to about 60 mg/kg/day. 27. A use according to claim 25 wherein the medicament is formulated to administer CMT in an amount from about 1 mg/kg/day to about 18 mg/kg/day. 28. A use according to claim 15 wherein the medicament is formulated for topical administration. 29. A use according to claim 28 wherein the medicament is formulated for administration as a mouthwash. 30. A method as claimed in claim 1 substantially as herein described with reference to any example thereof. 31. A use as claimed in claim 15 substantially as herein described with reference to any example thereof. j intellectual PROPERTY OPPICF OF N.Z 2 4 FEB 2004 RECEIVED </div>